There are great similarity and homology in an extracellular matrix of a tissue and organ of a human body and many animals. A biological matrix material manufactured by decellularization of an allogeneic or xenogenic tissue and organ has been successfully used for the repair and restoration of human tissues in clinical medicine. The decellularized tissue and organ matrix is also widely used for various studies in tissue engineering and regenerative medicine, for example, removing original cell components of a tissue and organ of animals, and re-cellularizing and functionalizing the matrix of the tissue and organ having a three-dimensional tissue scaffold structure by human cells in vitro, thereby finally producing the tissue and organ which can be implanted to human body.
The matrix of the tissue and organ is a three-dimensional scaffold composed of various complex structural proteins and functional proteins, and comprises many other active complexes. Main components include collagenous fiber, glycoprotein, mucoprotein, and the like, and the other components include saccharides such as glycosaminoglycan (hyaluronic acid, chondroitin sulfate), some lipids and growth factors. A good matrix of the tissue and organ has suitable biomechanical strength, and after being implanted into a host, the matrix material provides initial biomechanical support, and regulates cell behavior (e.g., adherence, migration, proliferation and differentiation) by interacting with a host cell, and the matrix of the tissue and organ itself is gradually degraded and converted into a new tissue with the ingrowth of the host cell.
Currently, there are approximately thirty kinds of matrix material products derived from tissues and organs all over the world, which have been used in various clinical medicines such as tissue repair and regeneration. Raw materials of the tissues and organs in these products are derived from tissues of human and various mammals, including blood vessel, cardiac valve, ligament, nerve, skin, small intestinal mucosa, forestomach, pericardium, peritoneum, muscle tendon and bladder, and the like.
A process procedure for manufacturing the matrix of the tissue and organ is very complex, including processes such as collection, preservation, washing, disinfection, decellularization, antigenicity reduction, virus inactivation, and terminal sterilization of the tissue and organ, and the like. There are many existing methods for manufacturing the matrix of the tissue and organ, and they can be classified into a physical method, a chemical method, an enzymatic method and the like according to their action principles of the decellularization. The most commonly used decellularization method is a method in which the physical treatment and chemical treatment are combined. The cellular membrane is damaged by stirring or ultrasonic, mechanical massage or pressurization, freezing and thawing, so that the cell components are released from the cell, further facilitating the subsequent decellularization and washing using a chemical detergent. The physical method itself is generally not sufficient to achieve complete decellularization. Enzymatic treatment method, such as some trypsin, can alter the density and porosity of the tissue extracellular matrix, and cut the connection between the cell surface and the tissue extracellular matrix. In addition, by using different process procedures and methods, the removal efficiency of cells and the effect or damage to the matrix of the tissue and organ are different. In addition to direct damage to the matrix of the tissue and organ, the collection, preservation, washing, disinfection, and decellularization treatment also influence the subsequent processing steps of the tissue and organ. Various treatments will influence and change the biochemical composition of the matrix of the tissue and organ, and the ultrastructure and biomechanical property of the three-dimensional scaffold to different extent, which will influence the response of the host to the implanted matrix material. The evidence of preclinical animal tests and human clinical application demonstrates that there are great differences among various products of the matrix of the tissue and organ in terms of the clinical performance of tissue repair and regeneration. The variation of characteristics of the matrix of the tissue and organ during the manufacture process is one of the main reasons causing the difference of clinical effects of various products.
Content of the Invention
In one aspect, the present invention provides a method for manufacturing an animal acellular tissue matrix material, which comprises the steps of:
(1). collecting a raw material of an animal tissue, wherein the animal tissue is washed to remove blood and other dirt, and cut into a tissue material having a length, a width and a height of the desired specification and dimension, and then the tissue material is preserved at a low temperature;
(2). thawing slowly, and rehydrating the tissue material in a normal saline containing gentamicin;
(3). disinfecting and sterilizing the tissue material in a moderate alkaline solution, wherein the tissue material is then rinsed with sterile pure water, and the pH of the tissue material is adjusted to be neutral;
(4). decellularizing and washing the tissue material;
(5). digesting DNA components of the animal tissue, wherein the animal tissue is then rinsed with a normal saline;
(6). digesting α-Gal antigen of the animal tissue, wherein the animal tissue is then rinsed with a high concentration of a sodium chloride solution, and rinsed with a normal saline;
(7). inactivating the viruses in the animal tissue the animal tissue, wherein the animal tissue is then rinsed with a phosphate buffer solution;
(8). packaging and sealing the animal tissue under an aseptic condition;
(9). terminal sterilization treatment.
In the method, an enzymatic method is used to remove cell components and α-Gal antigen and improve the pliability of a tissue scaffold.
In an embodiment of the present invention, the raw material of the animal tissue in step (1) is selected from skin, dermis, artery, vein, stomach, cartilage, meniscus, small intestine, large intestine, diaphragm, muscle tendon, ligament, nervous tissue, bladder, urethra and ureter.
In an embodiment of the present invention, the washing of the animal tissue to remove blood and dirt in step (1) is performed by using pure water and a physical method or ultrasonic washing.
In a process method for manufacturing the tissue matrix material, the step of preservation of a raw material of a porcine dermis at a low temperature is involved, wherein, the rate of cooling and heating is a very important parameter. If the rate of cooling and heating is too rapid or not uniform within the tissues, tiny cracks can occur in local regions of the tissues and the tissue matrix is prone to be tore when used.
In an embodiment of the present invention, preferably, the tissue material in step (1) is preserved at a temperature of −40° C. or less which is achieved with an average cooling rate of no more than 1.0° C. per minute, and more preferably, the cooling rate is 0.5° C. per minute. The tissue material preserved at a low temperature is slowly thawed in an environment of 5° C. to 12° C. in step (2), to avoid the production of cracks in the tissue due to an over-rapid temperature increase. After the ice is completely melted, the thawed tissue material is rehydrated in a normal saline containing 100 mg of gentamicin per liter for 3 hours to 6 hours in step (2).
In one embodiment of the present invention, the preservation at a low temperature in step (1) is long-term preservation, the method is to lay a porcine dermal material on a piece of protective layer with slightly larger area, such as cotton yarn cloth, paper, plastic film, nylon net or other cloth fabrics, and roll the dermis and the protective layer into one multilayer concentric roll or form a multilayer package form with the dermis and the protective layer being alternated, which is placed into a plastic bag, and kept in a refrigerator at −80° C. or −40° C. for preservation after being sealed.
In the preparation method of embodiments of the present invention, the initial disinfection and sterilization of a raw material of a porcine dermis are involved. The existing methods comprise use of sodium hypochlorite, peroxyacetic acid, hydrogen peroxide, iodine solution, and a high concentration of sodium hydroxide solution (with a pH of 13 or more). After the treatment using these solutions, the tissue matrix is damaged to different extent, especially with the effects of sodium hydroxide, sodium hypochlorite, and iodine solution being greater.
Unlike the disinfection and sterilization technology of the raw material in the existing methods, in an embodiment of the present invention, the moderate alkaline solution in step (3) is a sodium bicarbonate, or sodium hydroxide solution with a pH of 10.5 to 11.5 or an ammonia hydroxide solution with a concentration of 0.1%, the disinfection and sterilization method is to soak the rehydrated tissue material in the moderate alkaline solution for 24 hours to 48 hours with shaking slowly, thereby avoiding the damage of the tissue matrix.
In an embodiment of the present invention, the decellularization method in step (4) is to firstly rinse the disinfected and sterilized and rinsed tissue material in a normal saline containing 2.0 mmol/L of calcium chloride, 2.0 mmol/L of magnesium chloride and 100 mg/L of gentamicin at room temperature for 1 hours to 3 hours, and then add a dispase solution to elute cells.
In one embodiment of the present invention, the dispase solution is a neutral dispase solution, which contains 1 mmol/L to 20 mmol/L of calcium chloride, 1 mmol/L to 20 mmol/L of magnesium chloride and 50 units/L to 400 units/L of dispase, and the method for eluting cells with the dispase solution is to soak the tissue material in the neutral dispase solution, at 37° C. for 24 hours to 36 hours with shaking slowly, and more preferably, the neutral dispase solution contains 2.0 mmol/L of calcium chloride, 2.0 mmol/L of magnesium chloride and 100 units/L to 200 units/L of dispase.
In one embodiment of the present invention, after completing the decellularization in step (4), the washing step is performed. The washing comprises washing with a first detergent and/or washing with a second detergent, wherein the first detergent solution is prepared by dissolving polyethylene glycol tert-octylphenyl ether Triton™ X-100, at a concentration of 0.5%, in a buffer solution of hydroxyethylpiperazine ethane sulfonic acid (pH 7.0˜8.0), and the washing method is to soak the tissue material in the first detergent solution at 37° C. for 12 hours to 18 hours with shaking slowly. The second detergent solution is prepared by dissolving sodium deoxycholate, at a concentration of 1.0%, in a phosphate buffer solution (pH 7.2˜7.8), and the washing method is to soak the tissue material in the second detergent solution at room temperature for 24 hours to 36 hours with shaking slowly. Meanwhile, other suitable detergents, such as Tween-20, t-octylphenoxyl polyethylene ethoxyethanol and 3-[(3-cholesterol aminopropyl)dimethylamino]-1-propanesulfonic acid, and the like, are used in embodiments of the present invention.
In an embodiment of the present invention, after being soaked in the first detergent solution and the second detergent solution, and prior to step (5), the tissue material is rinsed three times with a buffer solution of 20 mmol/L of hydroxyethylpiperazine ethane sulfonic acid (with a pH between 7.0˜8.0) at room temperature, each time for 2 hours to 4 hours.
Due to the existence of DNA of the animal tissues, an inflammatory response is easily caused by the tissue matrix being implanted into a human body. In addition to human and old world monkeys, other mammals all contain α-Gal antigen consisting of glycoprotein or glycolipid with a disaccharide end of α-1,3-galactose residue [Galα(1,3)Gal] in vivo. In particular, the α-Gal antigen in porcine tissues will cause immunological rejection response. One of the methods for eliminating or overcoming the inflammatory response and rejection response is to remove DNA and α-Gal antigen from the animal tissue matrix using specific enzymatic treatment.
In an embodiment of the present invention, the digestion of DNA components of the animal tissue in step (5) is accomplished by adding a deoxyribonuclease solution, wherein the deoxyribonuclease solution is prepared by adding 2.0 mmol/L of calcium chloride, 2.0 mmol/L of magnesium chloride and 5000 enzyme units/L of deoxyribonuclease into a buffer solution of 100 mmol/L of tri-hydroxymethyl aminomethane-hydrochloric acid with a pH of 7.2, and a method for digesting DNA from the animal tissue is to soak the tissue material in the deoxyribonuclease solution to be treated for 18 hours to 28 hours at 37° C. with shaking slowly, and then place the tissue material in a normal saline to be rinsed twice at room temperature, each time for 1 hours to 3 hours.
In an embodiment of the present invention, digesting α-Gal antigen of the animal tissue in step (6) is accomplished by adding α-galactosidase solution, wherein the formula of the α-galactosidase solution is to add 2.0 mM calcium chloride, 2.0 mM magnesium chloride and 400GALU units of α-galactosidase into per liter of a buffer solution of 10 mM hydroxyethylpiperazine ethane sulfonic acid (with a pH between 7.0˜8.0), and a method for digesting the α-Gal antigen of the animal tissue is to soak the tissue material in the α-galactosidase solution, to be washed for 24˜36 hours at 37° C. with shaking slowly.
When the tissue matrix implanted into human body is manufactured, it is necessary to remove various residual enzymes. To achieve the above objectives, in an embodiment of the present invention, the washing is performed using a salting-out method, wherein the high concentration of sodium chloride solution in step (6) is a 2 to 5% sodium chloride solution, and a rinsing method is to soak the tissue material in the sodium chloride solution and wash the tissue material twice at room temperature, each time for 2 hours to 4 hours. In one-embodiment, the high concentration of sodium chloride solution can be a 3% sodium chloride solution. Furthermore, the sodium chloride solution can be replaced with other neutral salt solution, such as potassium chloride, magnesium chloride and lithium chloride, and the like.
To increase the safety of the products, in an embodiment of the present invention, said method also relates to a virus inactivation treatment, wherein the virus inactivation agents used in step (7) are hydrogen peroxide and peroxyacetic acid, and a method for the virus inactivation is to soak the tissue material in a solution containing 0.01% to 0.10% hydrogen peroxide, 0.05% to 0.50% acetic acid and 0.05% to 0.50% peroxyacetic acid, to be washed for 2 hours to 3 hours at room temperature with shaking slowly. In another embodiment of the present invention, a solution containing 0.02% hydrogen peroxide, 0.15% acetic acid and 0.10% peroxyacetic acid is used for virus inactivation, with the number of viruses being decreased by 106 or more during 2 hours to 3 hours. The concentration of hydrogen peroxide, acetic acid and peroxyacetic acid may be varied with the number of bacteria.
In an embodiment of the present invention, after the virus inactivation in step (7), the tissue material is rinsed three times at room temperature with a neutral phosphate buffer solution, each time for 2 hours to 4 hours, to remove the residual hydrogen peroxide, acetic acid and peroxyacetic acid.
The terminal sterilization treatment of the tissue product is often one of the most destructive steps for the tissue material. For this reason, in an embodiment of the present invention, a low temperature irradiation is used to perform the treatment in step (9). In another embodiment of the present invention, under the condition of −40° C., the terminal sterilization treatment of the tissue material is performed using 10 kGy to 50 kGy gamma ray, which greatly reduces the damage to the tissue material. In some embodiments of the present invention, a radiation dosage is varied depending on the number of bacteria in the tissue matrix. In another embodiment, the terminal sterilization treatment of the tissue material is performed using 20 kGy to 30 kGy gamma ray.
In some alternative embodiments of the present invention, in addition to the irradiation terminal sterilization method, the tissue matrix in said method can also be sterilized by using ethylene oxide gas after being lyophilized.
In some embodiments of the present invention, the sequence of step (4) (decellularization with enzyme), step (5) (digesting DNA with enzyme) and step (6) (digesting α-1,3-galactose residue antigen with enzyme) can be adjusted or altered as actually required. For example, firstly, α-1,3-galactose residue antigen in animal tissues may be digested, and then the animal tissues are decellularized and the DNA components of animals are digested; or firstly, the animal DNA is eliminated, and then the α-1,3-galactose residue antigen is eliminated and finally treated by decellularization.
Furthermore, in some alternative embodiments of the present invention, if the animals improved by genetic engineering and free of α-Gal antigen are selected, step (6) is omitted and step (7) is directly performed. Meanwhile, to reduce the disadvantageous effect on the proteolysis of the extracellular matrix, the concentration of the dispase, temperature and time will be monitored and optimized while treating. In the process procedure, the specific enzyme inhibitor can further be added, for example, ethylenediamine tetraacetic acid, for inhibiting the activity of the dispase.
Another aspect of the present invention further relates to an animal acellular tissue matrix material manufactured by the above method of the embodiments of the present invention.
In one embodiment of the present invention, said animal acellular tissue matrix material is obtained by using dermis with basement membrane or dermis with basement membrane removed as a raw material of an animal tissue.
In the method for manufacturing an acellular tissue matrix material according to the embodiments of the present invention, a series of steps of treating animal skin tissues and manufacturing the matrix of the tissue and organ as well as a plurality of biochemical solutions and formulas thereof are involved. The dermal tissue matrix material manufactured by the above steps and solutions retains the original basic scaffold structure of the tissue extracellular matrix, main biochemical components and biomechanical strength, with an antigen causing immunological rejection response in the human body being effectively removed from the animal tissue; and improves the flexibility, drapability and the integration performance of wound curved surface of the tissue matrix, and the manufactured animal dermis matrix is similar with human skin, which will not cause the collagen in the tissue matrix to crosslink with other proteins, and will not cause degradation or denaturation, and the decellularized dermis tissue retains the biological integrity of the natural dermal tissue matrix.